The desirability of using entomogenous nematodes in compositions to control pests in agricultural contexts has been clearly recognized. The general classification "nematodes" includes roundworms of astounding variety ranging in size from ultramicroscopic to worms of several feet in length. In many cases, these organisms are parasitic, and their mechanisms of parasitism and their targets are as varied as their size range. Plant pathogenic nematodes are a recognized agricultural nuisance, and various toxic compounds have been formulated to control their growth. Parasitic diseases in animals and humans caused by these parasites include heartworm in dogs and river blindness in humans.
However, a relatively benevolent group of nematodes infects insects that are themselves agricultural pests, and thus this group is seen as having a useful function. In particular, control of insect infestation of plants has been attempted using the "infective juvenile" stage of these entomogenous nematodes. In this form, the infective juvenile or "IJ" enters the host insect through the alimentary canal or spiracles, emerges from its protective sheath, and penetrates into the host insect's hemocoel. In the host insect's hemocoel the nematode releases symbiotic bacteria which induce septicemia that kills the host and renders the host corpse suitable for nematode foraging and reproduction. Several generations may be spent within the insect host until food consumption and overpopulation trigger production of another IJ stage generation. The new infective juveniles must then find fresh hosts.
In the infective juvenile stage, the nematodes do not eat, but depend upon internal food stores; however, they do require oxygen, and, unless induced into a cryptobiotic desiccated state, require the presence of water.
A major problem associated with the use of infective juvenile nematodes as pesticides resides in the necessity to maintain the infective juveniles in a viable state for extended time periods. The infective juveniles cannot be kept viable simply by harvesting them from, for example, an artificial culture, and placing them in a container. The majority of the infective juveniles so prepared will die within a few hours. Various attempts have been reported to overcome this problem; these attempts fall generally into two categories. In one approach a "cryptobiotic" state is induced by dehydration or other means so as to reduce the metabolism to the point where the infective juvenile essentially becomes inert. It is known that nematodes in the soil may exist in such a cryptobiotic state under dry climatic conditions; however, the statistics of this process are not favorable to preserving large numbers of infective juveniles. Alternative drying means which result in a cryptobiotic state with some degree of efficiency are described, for example, in European Patent Application published as Publication No. 256,873.
In an alternative approach, the IJs are stored on various moist, high surface area materials. For example, U.S. Pat. No. 4,417,545 describes a method and package for storage and shipment of nematodes which comprises essentially a foam sandwich wherein the foam retains water. While the container is stated to be "suitable for storing nematodes", only nematode eggs were tested, and only one particular foam was found to be successful in maintaining the viability of the eggs, even when storage was at the low temperature of -5.degree. C.
U.S. Pat. No. 4,765,275 also describes a packaging system for nematode storage and transport. In this proposal, the nematodes are stored as a suspension in a manner so as to prevent microbial growth (such as by adding formaldehyde to the suspension) and in the presence of an adsorbent, such as activated charcoal.
U.S. Pat. No. 4,615,883 describes a formulation of infectious juveniles in which the IJs are encapsulated in an alginate gel obtained by adding calcium ion to a sodium alginate suspension of the nematodes. In these preparations, capsules of 0.5-5 mm in diameter are formed and are said to improve the storageability of the IJs. The use of alginate gels and other encapsulating materials to encapsulate living tissue or cells (though not specifically IJs) has an extensive literature. See, for example, U.S. Pat. Nos. 4,409,331; 4,407,957; 4,391,909; 4,352,883; 4,663,286; 4,778,749; 4,798,786; 4,803,168; 4,806,355; 4,647,536; and 4,814,274. All of these patents describe methods to encapsulate living cells or tissues in various polymeric capsules. The purpose of the capsules in each case is to preserve the viability of the living tissue and, also, to facilitate the use of such tissue in its intended application.
U.S. application Ser. No. 07/313,594, filed Feb. 21, 1989, assigned to the same assignee and incorporated herein by reference, describes a packaging method for IJs in a reversibly cross-linked matrix which is embedded in a supporting screen. The screen containing the sheet matrix is simply placed in the location of end use, and application of water and an active decross-linking agent liberates the embedded nematodes from the film.
It has now been found that a more convenient and effective manner of preserving the viability of infective juveniles resides in immobilizing the IJs in a pseudoplastic gel, which can then readily be dispensed in the field by applying a suitable shear force. While embedded in the matrix, the IJs are immobilized, resulting in reduced metabolism and improved viability. However, upon liquefaction of the supporting medium, the IJs can be readily dispensed in the desired location.